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WO2022221367A1 - Production non endogène de cannabinoïdes et de composés précurseurs de cannabinoïdes dans des systèmes végétaux - Google Patents

Production non endogène de cannabinoïdes et de composés précurseurs de cannabinoïdes dans des systèmes végétaux Download PDF

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WO2022221367A1
WO2022221367A1 PCT/US2022/024548 US2022024548W WO2022221367A1 WO 2022221367 A1 WO2022221367 A1 WO 2022221367A1 US 2022024548 W US2022024548 W US 2022024548W WO 2022221367 A1 WO2022221367 A1 WO 2022221367A1
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heterologous
fragment
variant
plant
seq
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Simon KALMUS
Brian S. DEDECKER
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University of Colorado System
University of Colorado Denver
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University of Colorado System
University of Colorado Denver
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Definitions

  • the present invention relates to the heterologous production of therapeutically and commercially relevant compounds in transgenic plants.
  • the present invention relates to novel systems, methods and compositions for the production of cannabinoid compounds and their precursors in transgenic plants, namely soybeans.
  • CBD cannabidiol
  • transgenic yeast requires the use of expensive bioreactors, intensive sterilization procedures, and remains limited by expensive separation and purification techniques.
  • due to genetics it is difficult to produce appreciable quantities of these compounds due to competition for intermediates among specific cannabinoid pathways.
  • the present invention relates to the heterologous production of therapeutically and commercially relevant compounds in transgenic plants.
  • the present invention relates to novel systems, methods and compositions for the production of cannabinoids, such as cannabidiol (CBD) in transgenic plants, namely soybeans.
  • CBD cannabidiol
  • This method could also be used in seeds harvested from other commonly farmed crops such as corn, rice and rapeseeds.
  • the present invention relates to novel systems, methods and compositions for the production of cannabinoid intermediates and precursors in transgenic plants, namely soybeans.
  • the present invention relates to the inducible heterologous production of therapeutically and commercially relevant compounds in transgenic plants.
  • the present invention relates to novel systems, methods and compositions for the inducible production of cannabinoids, such as CBD in transgenic plants, namely soybeans seeds.
  • the present invention relates to novel systems, methods and compositions for the inducible production of cannabinoid intermediates and precursors and other compounds in transgenic plants, namely soybeans.
  • transgenic plant or seed of the invention may include a plant or seed, and preferably a soybean plant or seed, expressing a heterologous nucleotide sequence encoding one or more heterologous enzymes necessary for the production a cannabinoid, or a cannabinoid intermediate from a precursor compound, which may include one or more aldehydes.
  • the genes encoding the non-endogenous cannabinoid biosynthesis pathway may be subject to an inducible promoter, such as an AlcA/AlcR inducible expression system.
  • the non-endogenous cannabinoid biosynthesis pathway may be encoded and expressed in a plant, and preferably a soybean plant, by a heterologous nucleotide sequence comprising one or more expression cassettes, operably linked to promoter(s), encoding one or more of the following heterologous enzymes: a heterologous Aldehyde Dehydrogenase enzyme, a heterologous Hexanoyl-CoA Synthetase enzyme, a heterologous Olivetolic Acid Synthase, a heterologous Olivetolic acid cyclase, a heterologous Prenyl Transferase, preferably including cannabigerolic acid (CBGA) synthase; and a heterologous CBDA synthase enzyme, or a heterologous THCA synthase enzyme.
  • a heterologous Aldehyde Dehydrogenase enzyme a heterologous Hexanoyl-CoA Synthetase enzyme
  • a cannabinoid synthase such as CBDA synthase or the THCA synthase are coupled with localization signal forming a fusion peptide according to SEQ ID NO. 15 and 16, respectively, or a fragment or variant thereof.
  • This localization signal may preferably be configured to direct cannabinoid biosynthesis in soybean plant, and may include a Translocon on the inner chloroplast envelope protein 22 TIC(22) localization signal, or a fragment or variant thereof.
  • a transgenic seed of the invention may be contacted with a precursor such that the precursor is imbibed by the seed at levels greater than that of the wild-type level of said precursor.
  • a transgenic seed of the invention may be contacted with an aldehyde precursor, and preferably hexanal, butyraldehyde or octanal.
  • the exemplary precursor hexanal is incorporated into the non-endogenous cannabinoid pathway by the enzymatic action of aldehyde dehydrogenase forming hexanoic acid.
  • the non-endogenous cannabinoid biosynthesis pathway is thus driven to produce one or more cannabinoid intermediates, Hexanoyl-CoA, 3,5,7-Trioxododecanoyl-CoA; and Olivetolic acid (OA), or a combination of the same.
  • the non-endogenous cannabinoid pathway may use a non-endogenous aldehyde precursor, such as hexanal, butyraldehyde or octanal that may be incorporated into the cannabinoid biosynthesis pathway forming one or more of the following cannabinoids: cannabidiolic acid (CBDA); cannabidiol (CBD);tetrahydrocannabinolic acid (THCA); tetrahydrocannabinol (THC); cannabidivarinic acid (CBDVA); cannabidivarin (CBDV); cannabidiphorol (CBDP); tetrahydrocannabivarin (THCV); and/or tetrahydrocannabiphorol (THCP).
  • CBDDA cannabidiolic acid
  • CBD cannabidiol
  • THCA tetrahydrocannabinolic acid
  • THC tetrahydrocannabinol
  • FIG. 1 Method for production of compounds such as cannabinoids using transgenic plants.
  • Transgenic plants are grown and harvested with no concurrent production of CBD. This eliminates the problem of developmental defects that would be caused to the plant by shunting endogenous metabolites to the production of exogenous compounds such as CBD. In addition, the local environment and water supply will not be potentially contaminated with compounds such as CBD.
  • the harvested seeds can then be either stored for future production or transported immediately to a secure facility where they are imbibed and activated.
  • Biosynthesis can be activated by one of two ways; either by addition of chemical pathway intermediates to the incubating the bean in a precursor compound, such as hexanal for the production of CBD and other cannabinoids, or by activating the expression of the transgenes by using an inducible system such as the AlcA/AlcR inducible expression system. After adequate incubation during which compound synthesis takes place, the biomass is processed. The compound of interest is then extracted from the processed biomass and refined.
  • a precursor compound such as hexanal for the production of CBD and other cannabinoids
  • Figure 2 Design of the pathway for use of hexanal during seed germination for the biosynthesis of cannabidiol (CBD A).
  • CBD A cannabidiol
  • Chemical precursors may be fed to the germinating seeds for incorporation into the nonendogenous compound synthesis pathway.
  • hexanal is fed to the germinating seeds to be incorporated into the CBD synthesis pathway.
  • Feeding of the chemical precursor may also function as the inducer of the biosynthesis pathway during seed germination.
  • a series of 6 transgenes of the CBD biosynthesis pathway then utilize the added hexanal and endogenous chemical intermediates of the plant to synthesize CBD.
  • Aldehyde dehydrogenase (ALDH) is used to convert the hexanal to hexanoic acid.
  • ADH Aldehyde dehydrogenase
  • Acyl- Activating Enzyme (AAEl)activates the hexanoic acid via Coenzyme A ligation, forming Hexanoyl-CoA.
  • the enzyme Olivetolic Acid Synthase (OLS) incorporates 3 endogenous Malonyl-CoA groups to form 3,5,7-Trioxododecanoyl-CoA, OLS then works in conjunction with OAC to cyclize the molecule forming olivetolic acid (OA).
  • CBDAS Cannabigerolic Acid Synthase
  • GPP Geranyl Pyrophosphate
  • CBDA Cannabigerolic acid
  • CBDAS cannabidiolic acid synthase
  • Other precursors could be fed to the germinating seeds to make related compounds. For instance, butyraldehyde or octanal could be fed into the same pathway to produce CBDVA or CBDPA.
  • FIG. 3 Production of CBD in Soybean Tissue. Production of CBD in germinating soybean seeds of cultivar Williams 82. The seeds were infected with a DNA construct containing the CBD pathway genes shown in Figure 2 via agrobacterium strain EHA101. The chemical precursor hexanal was fed to the germinating seeds and the nonendogenous CBD pathway genes were induced with 0.4% EtOH via the AlcR/AlcA ethanol inducible expression system. The germinating seeds were incubated for 16 days on Gamborg B5 Medium. After the incubation, samples of the germinating seeds were homogenized via maceration. Organic compounds present in the sample were extracted with methanol and analyzed by triple quadrupole mass spectrometry. CBD production was detected in the germinating soybeans while other cannabinoids such as THC were not present.
  • FIG. 4 Control. Germinating soybean seeds were transfected as Figure 3, but with a control DNA plasmid that does not contain the CBD pathway genes. The germinating seeds were fed hexanal and mock induced with ethanal as Figure 3. Methanal extraction and analysis by triple quadrupole mass spectrometry showed no detectable CBD or any other cannabinoids. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention includes novel systems, methods, and compositions for the non- endogenous production of cannabinoids in a non -Cannabis plant.
  • the present invention includes novel systems, methods, and compositions for the non-endogenous production of cannabinoids in a non -Cannabis plant, namely soybean seeds.
  • An exemplary current transgenic plant would produce cannabinoids using soybeans that are amenable to modem farming practices and that allow for the more efficient and less expensive production of cannabinoids due to economies of scale. Soybean cultivation is much less expensive and more environmentally sustainable than the cultivation of cannabis which is both labor and resource intensive.
  • CBD and other cannabinoids are labor intensive to harvest and is often produced using indoor growing facilities that require intensive energy and land use. Extracting pure cannabinoids such as CBD and other minor cannabinoids from harvested cannabis plants is also expensive.
  • the synthesized hydrophobic cannabinoids from the seeds of the transgenic plant are readily solubilized by the natural oil present in the soybean.
  • the cannabinoid infused oils may then be easily extracted using current commercial methods for isolating oil from crop seeds. This is usually done by using expeller pressing or solvent-based extraction. Because the soybeans are incubated in a controlled environment, it is possible to add key intermediates for uptake by the soybean.
  • hexanal was added to the germinating soybeans in this example to produce CBD.
  • This can be applied for example to the biosynthesis other cannabinoids, cannabinoid intermediates and cannabinoid precursor compounds.
  • transformed seeds could be butyraldehyde were added to the system, then the expected product of the same pathway described for CBDA (Figure 2) would be CBDVA.
  • THC tetrahydrocannabinol acid
  • CBD produced in the transgenic soybean would be specific to the plant in regard to other cannabinoids. For instance, having no THCA synthase enzyme present, the transgenic plant would produce no THC. Naturally, the inverse could also be accomplished if THCA synthase were expressed in the transgenic seed producing THCA, and not CBDA.
  • the present invention method mitigates these problems, as the compound is produced in seeds that have already been harvested from the fully matured plant ( Figure 1).
  • the transgenic plant does not produce a non-endogenous cannabinoid, such as THC or CBD during development and growth of the plant in the field.
  • the seeds of the transgenic plant are harvested and transported to a production facility.
  • the seeds are germinated in a controlled environment and the synthesis of non-endogenous cannabinoid, such as THC or CBD is then initiated with an inducible system.
  • This allows the engineered crop to not produce non-endogenous cannabinoid, such as THC or CBD, their intermediates or precursors, as well as the associated enzymes while growing in the field, thereby allowing for farming of the crop without developmental defects or potentially contaminating the local environment and water supply with synthesized compounds (Figure 1).
  • Wild type (non-engineered) seeds may also be used in this method by transfecting them with the transgenic DNA after germination. For instance, the wild type seeds are harvested and germinated. Once germinated, the seeds are transfected with the transgenic DNA via the standard agrobacterium mediated method. From there, the biosynthesis pathway is activated to produce compounds.
  • Known inducible promoter systems have been developed for plants such as the ethanol inducible expression system from Aspergillus Nidulans (AlcR/AlcA).
  • the Aspergillus Nidulans AlcR/AlcA ethanol inducible expression system described in WO2001009357A2, by Syngenta Ltd., (incorporated herein by reference) can be used to induce expression of the synthetic metabolic pathway shortly after seed germination.
  • Other examples of the method may utilize a similar inducible promoter system such as the estradiol dependent XVE system to control the timing of recombinant gene expression.
  • the biosynthesis pathway in the germinating beans may also be induced by adding an essential chemical pathway precursor to the system.
  • the enzymes of the pathway are constitutively expressed, but the compound synthesis will not begin until a key chemical pathway precursor is added to the germinating seeds.
  • all the enzymes of the CBDA pathway would be constitutively expressed except those needed to produce the key chemical precursor hexanal. Once hexanal is added to the germinating seeds, the biosynthesis pathway will then proceed and begin producing CBDA.
  • Figure 2 displays the pathway that has been utilized in producing the cannabinoid CBD, and precursor for other cannabinoids such as CBD, in germinating soybean seeds.
  • CBDV could be produced by this same nonendogenous pathway by feeding butyraldehyde instead of hexanal to the germinating seeds.
  • Additional embodiments may include utilizing the systems and methods of the invention for the non-endogenous production of therapeutically and commercially relevant compounds found in a variety of other plants.
  • hundreds of compounds called terpenes from the MEV and MEP pathways of plant metabolism are highly relevant to a vast array of modern industries, such as the cosmetic, pharmaceutical, and food industries, among others. However, they often exist in trace quantities or are difficult to extract due to limitations to cultivation or production.
  • Additional embodiment may include utilizing the systems and methods of the invention for the nonendogenous production of other terpenes and alkaloids.
  • the present invention includes systems, methods and compositions to produce a transgenic plant having a non-endogenous cannabinoid biosynthesis pathway.
  • the transgenic plant or seed of the invention may include a plant or seed, and preferably a soybean plant or seed, expressing a heterologous nucleotide sequence encoding one or more heterologous enzymes necessary for the production a cannabinoid, or a cannabinoid intermediate from a precursor compound, which may include one or more aldehydes.
  • the genes encoding the non-endogenous cannabinoid biosynthesis pathway may be subject to an inducible promoter, such as an AlcA/AlcR inducible expression system according to SEQ ID NO.
  • the non-endogenous cannabinoid biosynthesis pathway may be encoded and expressed in a plant, and preferably a soybean plant, by a heterologous nucleotide sequence comprising an expression cassette, operably linked to a promoter, encoding one or more of the following heterologous enzymes: a heterologous Aldehyde Dehydrogenase enzyme, a heterologous Acyl-Activating enzyme, a heterologous Olivetolic Acid Synthase, a heterologous Olivetolic acid cyclase, a heterologous Prenyl Transferase comprising cannabigerolic acid (CBGA) synthase; and a heterologous CBDA synthase enzyme, or a heterologous THCA synthase enzyme., or , or fragments or variants of the same.
  • a heterologous nucleotide sequence comprising an expression cassette, operably linked to a promoter, encoding one or more of the following heterologous enzymes:
  • the non- endogenous cannabinoid biosynthesis pathway may be encoded and expressed in a plant, and preferably a soybean plant, by a heterologous nucleotide sequence comprising an expression cassette, operably linked to a promoter, encoding one or more of the following heterologous enzymes: a heterologous amino acid sequence according to SEQ ID NO. 4, a heterologous amino acid sequence according to SEQ ID NO. 5, a heterologous amino acid sequence according to SEQ ID NO. 6, a heterologous amino acid sequence according to SEQ ID NO. 7, a heterologous amino acid sequence according to SEQ ID NO. 8; and a heterologous amino acid sequence according to ID NO. 9 or 14.
  • Another embodiment of the invention includes the localized production of cannabinoid and cannabinoid intermediates from a non-endogenous cannabinoid biosynthesis pathway.
  • a cannabinoid synthase such as CBDA synthase or the THCA synthase are coupled with localization signal forming a fusion peptide.
  • This localization signal may preferably be configured to direct cannabinoid biosynthesis in soybean plant, and may include a TIC22 localization signal according to SEQ ID NO. 22, or a fragment or variant thereof.
  • the present invention includes systems, methods and compositions for the non-endogenous production of cannabinoids and cannabinoid intermediates in a plant seed.
  • the invention may include a system for transforming a plant, and preferably a soybean plant to produce a transgenic seed expresses a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more heterologous enzymes necessary for the production a cannabinoid, or a cannabinoid intermediate from a precursor compound.
  • the non-endogenous pathway may be activated through contacting the transgenic seed with a quantity of a cannabinoid precursor compound, such as an aldehyde, which can be incorporated into the non-endogenous pathway.
  • a transgenic seed of the invention may be contacted with a precursor such that the precursor is imbibed by the seed at levels greater than that of the wild-type level of said precursor.
  • a transgenic seed of the invention may be contacted with an aldehyde precursor, and preferably hexanal or butyraldehyde.
  • the exemplary precursor hexanal is incorporated into the non-endogenous cannabinoid pathway by the enzymatic action of aldehyde dehydrogenase forming hexanoic acid.
  • the non-endogenous cannabinoid biosynthesis pathway is thus driven to produce one or more cannabinoid intermediates, Hexanoyl- CoA, 3,5,7-Trioxododecanoyl-CoA; and Olivetolic acid (OA).
  • cannabinoid intermediates Hexanoyl- CoA, 3,5,7-Trioxododecanoyl-CoA; and Olivetolic acid (OA).
  • the OA produced by the non-endogenous cannabinoid biosynthesis pathway may be converted into a cannabinoid intermediate by a prenyl transferase, in this case CBGA synthase (SEQ ID NO. 8) to form CBGA.
  • CBGA synthase SEQ ID NO. 8
  • the CBGA produced non-endogenous cannabinoid biosynthesis pathway of the invention may further be converted into one or more cannabinoids through the enzymatic action of a cannabinoid synthase.
  • a heterologous CBDA synthase enzyme according to amino acid sequence SEQ ID NO. 9 or a heterologous THCA synthase enzyme according to amino acid sequence SEQ ID NO. 14, or a fragment or variant thereof.
  • the present invention includes systems, methods and compositions for the non-endogenous production of cannabinoids and cannabinoid intermediates.
  • the invention may include the step of transforming a non -Cannabis plant, and preferably a soybean plant or other high-biomass crop/plant, to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more heterologous enzymes necessary for the production a cannabinoid, or a cannabinoid intermediate from a precursor compound.
  • Non-endogenous cannabinoid biosynthesis pathway may be induced, for example by inducing the promoter, or contacting a precursor to the plant system such that the precursor is incorporated into the non-endogenous pathway.
  • the resulting cannabinoids, and cannabinoid intermediate may be isolated from the plant biomass and further purified for commercial, therapeutic, or recreational use.
  • the non-endogenously produced cannabinoid of the invention may include cannabidiolic acid (CBDA), cannabidiol (CBD), tetrahydrocannabinolic acid, cannabidivarinic acid (CBDVA), or cannabidivarin (CBDV), cannabidiphorolic acid (CBDPA), cannabidiphorol (CBDP), tetrahydrocannabinolic acid (THCA), tetrahydrocannabinol (THC), tetrahydrocannabivarin acid (THCVA), tetrahydrocannabivarin (THCV), tetrahydrocannabiphorolic acid (THCPA), tetrahydrocannabiphorol (THCP) and a cannabinoid intermediate of the invention may include cannabigerolic acid (CBGA) or cannabigerol (CBG).
  • CBDA cannabigerol
  • CBD cannabig
  • the present invention includes systems, methods and compositions for the non-endogenous production of precursors for cannabinoids and cannabinoid intermediates.
  • transgenic plant may be configured to express a heterologous nucleotide sequence, operably linked to a promoter, encoding one or more heterologous enzymes necessary for the production a one or more of cannabinoid precursors, which may include: Hexanoic acid, Hexanoyl-CoA, 3,5,7-Trioxododecanoyl-CoA; and Olivetolic acid (OA), or a combination of the same.
  • Hexanoic acid Hexanoyl-CoA
  • 3,5,7-Trioxododecanoyl-CoA 3,5,7-Trioxododecanoyl-CoA
  • OA Olivetolic acid
  • Additional aspects of the invention include isolated nucleotide sequences and expression vectors encoding one or more enzymes necessary to non-endogenously produce a cannabinoid, cannabinoid intermediate, or cannabinoid precursor compounds, or a combination of the same.
  • the invention may include an expression vector having a nucleotide sequence, operably linked to a promoter, encoding one or more of the following: a heterologous Aldehyde Dehydrogenase enzyme; a heterologous Acyl -Activating enzyme; a heterologous Olivetolic Acid Synthase; a heterologous Olivetolic acid cyclase; a heterologous Prenyl Transferase comprising cannabigerolic acid (CBGA) synthase; and a heterologous CBDA synthase enzyme, or a heterologous THCA synthase enzyme, all of which may be part of a single or separate expression cassette.
  • a heterologous Aldehyde Dehydrogenase enzyme a heterologous Acyl -Activating enzyme
  • a heterologous Olivetolic Acid Synthase a heterologous Olivetolic acid cyclase
  • a heterologous Prenyl Transferase comprising can
  • a plant or seed may be transformed by one of the expression vectors of the invention producing one or more cannabinoid precursors, while optionally producing a cannabinoid intermediate or cannabinoid.
  • the cannabinoid precursors may be isolated from the biomass, or may be used as feedstock for a separate cannabinoid production system, such as an in vitro production system (PCT/IB2015/056445), a cannabinoid production bioreactor (PCT/IB2015/056445, incorporated herein by reference), a yeast-based cannabinoid production system (PCT/US2018/029668, incorporated herein by reference), or a bacterial-based production system (PCT/US2018/054400, incorporated herein by reference), where they may be further converted into a cannabinoid intermediate or cannabinoid.
  • PCT/IB2015/056445 in vitro production system
  • PCT/IB2015/056445 a cannabinoid production bioreactor
  • a “cannabinoid” is a chemical compound (such as cannabinol, THC or cannabidiol) that is found in the plant species Cannabis among others like: Echinacea ; Acmella Oleracea Helichrysum Umbraculigerum ; Radula Marginata (Liverwort) and Theobroma Cacao , and metabolites and synthetic analogues thereof that may or may not have psychoactive properties.
  • Cannabinoids therefore include (without limitation) compounds (such as THC) that have high affinity for the cannabinoid receptor (for example Ki ⁇ 250 nM), and compounds that do not have significant affinity for the cannabinoid receptor (such as cannabidiol, CBD).
  • Cannabinoids also include compounds that have a characteristic dibenzopyran ring structure (of the type seen in THC) and cannabinoids which do not possess a pyran ring (such as cannabidiol).
  • a partial list of cannabinoids includes THC, CBD, dimethyl heptylpentyl cannabidiol (DMHP-CBD), 6,12- dihydro-6-hydroxy-cannabidiol (described in U.S. Pat. No. 5,227,537, incorporated by reference); (3S,4R)-7-hydroxy- ⁇ 6-tetrahydrocannabinol homologs and derivatives described in U.S. Pat. No.
  • cannabinoids are tetrahydrocannabinol, cannabidiol, cannabigerol, cannabichromene, cannabicyclol, cannabivarin, cannabielsoin, cannabicitran, cannabigerolic acid, cannabigerolic acid monomethylether, cannabigerol monomethylether, cannabigerovarinic acid, cannabigerovarin, cannabichromenic acid, cannabichromevarinic acid, cannabichromevarin, cannabidolic acid, cannabidiol monomethylether, cannabidiol-C4, cannabidivarinic acid, cannabidiorcol, delta-9-tetrahydrocannabinolic acid A, delta-9- tetrahydrocannabinolic acid B, delta-9-tetrahydrocannabinolic acid-C4, delta-9- tetrahydrocannabivarinic acid,delta-9-
  • cannabinoids within the context of this disclosure include tetrahydrocannabinol and cannabidiol.
  • cannabinoid may also include different modified forms of a cannabinoid such as a hydroxylated cannabinoid or cannabinoid carboxylic acid.
  • a UGT were to be capable of glycosylating a cannabinoid, it would include the term cannabinoid as defined elsewhere, as well as the aforementioned modified forms. It may further include multiple glycosylation moieties.
  • an “intermediate cannabinoid” comprises a cannabinoid that can be further converted into another cannabinoid.
  • CBGA as an intermediate cannabinoid, can be converted into THCA or CBDA.
  • identification of the acidic form of a cannabinoid also explicitly includes the decarboxylated form as described here.
  • a “precursor” includes all chemical compounds along the biosynthetic pathway that that precede a cannabinoid or cannabinoid intermediate.
  • the aldehyde is any aldehyde made from a fatty acid or fatty acid derivative.
  • the R group is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbons in length.
  • a polypeptide can be expressed in monocot plants and/or dicot plants.
  • Techniques for introducing nucleic acids into plants are known in the art, and include, without limitation, Agrobacterium- mediated transformation, viral vector-mediated transformation, electroporation, and particle gun transformation (also referred to as biolistic transformation). See, for example, U.S. Pat. Nos. 5,538,880; 5,204,253; 6,329,571; and U.S. Pat. No. 6,013,863; Richards et al., Plant Cell. Rep. 20:48-20 54 (2001); Somleva et al., Crop Sci.
  • intergenic transformation of plastids can be used as a method of introducing a polynucleotide into a plant cell.
  • the method of introduction of a polynucleotide into a plant comprises chloroplast transformation.
  • the leaves and/or stems can be the target tissue of the introduced polynucleotide. If a cell or cultured tissue is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures if desired, by techniques known to those skilled in the art.
  • Suitable methods for introduce polynucleotides include electroporation of protoplasts, polyethylene glycol-mediated delivery of naked DNA into plant protoplasts, direct gene transformation through imbibition (e.g., introducing a polynucleotide to a dehydrated plant), transformation into protoplasts (which can comprise transferring a polynucleotide through osmotic or electric shocks), chemical transformation (which can comprise the use of a polybrene- spermidine composition), microinjection, pollen-tube pathway transformation (which can comprise delivery of a polynucleotide to the plant ovule), transformation via liposomes, shoot apex method of transformation (which can comprise introduction of a polynucleotide into the shoot and regeneration of the shoot), sonication-assisted agrobacterium transformation (SAAT) method of transformation, infiltration (which can comprise a floral dip, or injection by syringe into a particular part of the plant (e.g., leaf)), silicon-
  • homolog or “variant,” used with respect to an original enzyme or gene of a first family or species, refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs or variant will have functional, structural or genomic similarities. Techniques are known by which homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes. A “fragment” used with respect to an original enzyme or gene refers to a truncated portion of the peptide or gene that still retains its intended function.
  • operably linked when used in reference to a regulatory sequence and a coding sequence, means that the regulatory sequence affects the expression of the linked coding sequence.
  • Regulatory sequences or “control elements,” refer to nucleotide sequences that influence the timing and level/amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters; translation leader sequences; introns; enhancers; stem-loop structures; repressor binding sequences; termination sequences; polyadenylation recognition sequences; etc. Particular regulatory sequences may be located upstream and/or downstream of a coding sequence operably linked thereto. Also, particular regulatory sequences operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.
  • promoter refers to a region of DNA that may be upstream from the start of transcription, and that may be involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • a promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence which may be operably linked to a coding sequence for expression in a cell.
  • An “inducible” promoter may be a promoter which may be under environmental control. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters.
  • a “constitutive” promoter is a promoter which may be active under most environmental conditions or in most cell or tissue types.
  • transformation refers to the transfer of one or more nucleic acid molecule(s) into a cell.
  • a plant is “transformed” or “genetically modified” by a nucleic acid molecule transduced into the plant when the nucleic acid molecule becomes stably replicated by the plant.
  • transformation or “genetically modified” encompasses all techniques by which a nucleic acid molecule can be introduced into, such as a plant.
  • vector refers to some means by which DNA, RNA, a protein, or polypeptide can be introduced into a host.
  • the polynucleotides, protein, and polypeptide which are to be introduced into a host can be therapeutic or prophylactic in nature; can encode or be an antigen; or can be regulatory in nature, etc.
  • vectors including virus, plasmid, bacteriophages, cosmids, and bacteria.
  • An “expression vector” is nucleic acid capable of replicating in a selected host cell or organism.
  • An expression vector can replicate as an autonomous structure, or alternatively can integrate, in whole or in part, into the host cell chromosomes or the nucleic acids of an organelle, or it is used as a shuttle for delivering foreign DNA to cells, and thus replicate along with the host cell genome.
  • an expression vector are polynucleotides capable of replicating in a selected host cell, organelle, or organism, e.g., a plasmid, virus, artificial chromosome, nucleic acid fragment, and for which certain genes on the expression vector (including genes of interest) are transcribed and translated into a polypeptide or protein within the cell, organelle or organism; or any suitable construct known in the art, which comprises an “expression cassette.”
  • a “cassette” is a polynucleotide containing a section of an expression vector of this invention. The use of a cassette assists in the assembly of the expression vectors.
  • An expression vector is a replicon, such as plasmid, phage, virus, chimeric virus, or cosmid, and which contains the desired polynucleotide sequence operably linked to the expression control sequence(s).
  • nucleic acid molecules encoding the proteins and chimeras of the invention may be used in the design of nucleic acid molecules encoding the proteins and chimeras of the invention in order to optimize expression in a particular host cell system.
  • all nucleotides of the present invention may be optimized for expression in a select organisms such a Glycine Max.
  • a polynucleotide sequence is operably linked to an expression control sequence(s) (e.g., a promoter and, optionally, an enhancer) when the expression control sequence controls and regulates the transcription and/or translation of that polynucleotide sequence.
  • an expression control sequence e.g., a promoter and, optionally, an enhancer
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), the complementary (or complement) sequence, and the reverse complement sequence, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell.
  • oligonucleotides and polynucleotides that are not commercially available can be chemically synthesized e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), or using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res. 12:6159- 6168 (1984). Other methods for synthesizing oligonucleotides and polynucleotides are known in the art. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149 (1983).
  • plant or “plant system” includes whole plants, plant organs, progeny of whole plants or plant organs, embryos, somatic embryos, embryo-like structures, protocorms, protocorm- like bodies (PLBs), and culture and/or suspensions of plant cells.
  • Plant organs comprise, e.g., shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, trichomes and the like).
  • the invention may also include Glycine max plants or seeds.
  • expression refers to the process by which the coded information of a nucleic acid transcriptional unit (including, e.g., genomic DNA or cDNA) is converted into an operational, non- operational, or structural part of a cell, often including the synthesis of a protein.
  • Gene expression can be influenced by external signals; for example, exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene can also be regulated anywhere in the pathway from DNA to RNA to protein.
  • Gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they have been made, or by combinations thereof.
  • Gene expression can be measured at the RNA level or the protein level by any method known in the art, including, without limitation, Northern blot, RT-PCR, Western blot, or in vitro , in situ , or in vivo protein activity assay(s).
  • nucleic acid or “nucleic acid molecules” include single- and double-stranded forms of DNA; single- stranded forms of RNA; and double-stranded forms of RNA (dsRNA).
  • dsRNA double-stranded forms of RNA
  • nucleotide sequence or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex.
  • RNA is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA).
  • RNA is inclusive of iRNA (inhibitory RNA), dsRNA (double stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (micro-RNA), hpRNA (hairpin RNA), tRNA (transfer RNA), whether charged or discharged with a corresponding acetylated amino acid), and cRNA (complementary RNA).
  • deoxyribonucleic acid” (DNA) is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids.
  • nucleotide sequence and “nucleotide sequence segment,” or more generally “sequence,” will be understood by those in the art as a functional term that includes both genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that encoded or may be adapted to encode, peptides, polypeptides, or proteins.
  • gene refers to a coding region operably joined to appropriate regulatory sequences capable of regulating the expression of the gene product (e.g., a polypeptide or a functional RNA) in some manner.
  • a gene includes untranslated regulatory regions of DNA (e.g., promoters, enhancers, repressors, etc.) preceding (up-stream) and following (down-stream) the coding region (open reading frame, ORF) as well as, where applicable, intervening sequences (i.e., introns) between individual coding regions (i.e., exons).
  • structural gene as used herein is intended to mean a DNA sequence that is transcribed into mRNA which is then translated into a sequence of amino acids characteristic of a specific polypeptide. It should be noted that any reference to a SEQ ID, or sequence specifically encompasses that sequence, as well as all corresponding sequences that correspond to that first sequence. For example, for any amino acid sequence identified, the specific specifically includes all compatible nucleotide (DNA and RNA) sequences that give rise to that amino acid sequence or protein, and vice versa.
  • a nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.
  • Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications (e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moieties: for example, peptides; intercalators: for example, acridine, psoralen, etc.; chelators; alkylators; and modified linkages: for example, alpha anomeric nucleic acids, etc.).
  • intemucleotide modifications e.g., uncharged linkages: for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.; charged linkages: for example, phosphorothioates, phosphorodithioates, etc.; pendent moi
  • nucleic acid molecule also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hair-pinned, circular, and padlocked conformations.
  • sequence identity or “identity,” as used herein in the context of two nucleic acid or polypeptide sequences, refers to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • nucleic acid refers to a quantity, level, value, or amount that varies by as much as 30%, or in another embodiment by as much as 20%, and in a third embodiment by as much as 10% to a reference quantity, level, value or amount.
  • heterologous or exogenous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or is synthetically designed, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • host cell is meant a cell which contains an introduced nucleic acid construct and supports the replication and/or expression of the construct.
  • Example 1 Production of cannabidiol CBD through spatiotemporal control of recombinant genes
  • the present inventors have introduced transgenes into soybean seeds ( Glycine max) that are able to synthesize the small molecule compound cannabidiol (CBD) during germination.
  • the compound can be traditionally found in the Cannabis Sativa plant, while a transgenic soybean plant could produce large quantities of CBD for production purposes. This process is less expensive than labor-intensive and resource-heavy cannabis production and allows for the production of transgenic soybeans that are free of THC.
  • the invention includes a nucleotide expression construct that has been configured to inducible express the enzymes needed to synthesize the compound CBD in plants. Testing by mass spec has demonstrated that the construct successfully produces CBD in tissue samples from soybean. The method was shown to produce 400 picograms per gram of germinating seed.
  • an exemplary nonendogenous pathway for production of CBD of the inventions includes:
  • Aldehyde Dehydrogenase Hexanal was added to the headspace of the germinating soybean seeds. Once diffused into the seed, nonendogenous aldehyde dehydrogenase oxidizes the hexanal to form hexanoic acid.
  • Acyl-Activating Enzyme (AAE1) The next step is the addition of the endogenous compound Coenzyme A (CoA) to hexanoic acid to form Hexonyl-CoA.
  • CoA Coenzyme A
  • hexanoic acid Hexonyl-CoA.
  • the Acyl-Activating Enzyme from Cannabis sativa AAEl (SEQ ID NO. 5) is used for this addition reaction.
  • the next step is the sequential addition of three molecules of endogenous Malonyl-CoA to the Hexanoyl-CoA intermediate to form the unstable 3,5,7-Trioxododecanoyl-CoA intermediate.
  • the type III polyketide synthase Cannabis sativa OLS (SEQ ID NO. 6) is used for this addition reaction.
  • Olivetolic acid cyclase OAC: The next step is to cyclize the unstable 3,5,7- Trioxododecanoyl-CoA intermediate to form the final product of cannabidiol (CBD).
  • CBDC cannabidiol
  • SEQ ID NO. 7 The DABB protein Cannabis sativa CBDC (SEQ ID NO. 7) is used for this cyclization reaction.
  • CBGAS Prenyl Transferase
  • CBD A Synthase The final step is the production of cannabidiolic acid from the CBGA intermediate via Cannabidiolic Acid Synthase. (NCBI Accession number A6P6V9) The synthase was fused with the (TIC22) localization signal (SEQ ID NO. 9) on the N-terminus to target the enzyme to the soluble fraction of the chloroplast envelope.
  • THCA Synthase The final step is the production of THCA from the CBGA intermediate via THCA Synthase. (SEQ ID NO. 14)
  • the synthase may be fused with the TIC22 localization signal (SEQ ID NO. 1) on the N-terminus to target the enzyme to the soluble fraction of the chloroplast envelope.
  • Oxidation of CBD A and THCA Hexanal is known to induce the production of reactive oxygen species in the chloroplast and this environment likely decarboxyl ated the CBDA and THCA products forming CBD and THC respectively (figure 3) during the incubation of the germinating soybean seed. Plasmids were constructed using a modified Golden Braid technique in order to assemble the DNA sequences synthesized by Twist Biosystems before insertion into a modified agrobacterium binary plasmid.
  • All heterologous pathway protein coding sequences were driven the inducible AlcA/AlcR system with the AlcA promoter sequence according to SEQ ID NO. 2.
  • a 33 base pair spacer (SEQ ID NO. 3) was utilized between the transcription start site and translation start site for all 6 exogenous cannabinoid pathway enzymes (SEQ ID NO. 4-9) shown in Figure 2.
  • Terminators according to SEQ ID NO. 10-13 were used to comprise the 3’ untranslated regions (3’ UTR) of the transgenes.

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Abstract

La présente invention comprend de nouveaux systèmes, des procédés, et des compositions pour la production non endogène de cannabinoïdes dans des plantes non-Cannabis, et de préférence de la Glycine max ou d'autres cultures à biomasse élevée. Dans un mode de réalisation, la présente invention comprend la génération de graines de plantes transgéniques, et de préférence des graines de Glycine max, exprimant une séquence nucléotidique hétérologue codant pour une ou plusieurs enzymes hétérologues nécessaires à la production d'un cannabinoïde, un intermédiaire cannabinoïde à partir d'un composé précurseur. Certains précurseurs peuvent être produits de manière endogène par la plante, ou incorporés à partir d'une source hétérologue et incorporés dans la voie cannabinoïde non endogène.
PCT/US2022/024548 2021-04-13 2022-04-13 Production non endogène de cannabinoïdes et de composés précurseurs de cannabinoïdes dans des systèmes végétaux Ceased WO2022221367A1 (fr)

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